1. Harvest cells from a culture which is at or near peak density by centrifuging at 100 x g for 1 minute. Note: Centrifugation at the lowest speed and the shortest time to allow sedimentation of the cells will maximize recovery.
2. Adjust the concentration of cells to 4 x 106/ml with fresh broth medium.
3. Transfer the concentrated cell suspension to a sterile Petri dish and allow the cells to remain undisturbed for at least one hour.
4. Transfer the cell suspension (note the volume) from the Petri plate to a 15 ml plastic centrifuge tube.
5. Add an equal volume of 6% (v/v) sterile reagent grade methanol solution that has been prepared in fresh ATCC medium 351.
6. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation). The time from mixing of the cell preparation and the methanol solution, before the cooling cycle begins, should be no greater than 15 min.
7. Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1 C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)
8. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials can be stored between -80 and -70°C for no longer than one week.
9. To establish a culture from the frozen state follow steps 1-4 listed above under the heading ESTABLISHING A CULTURE FROM A FROZEN AMPULE.